Understanding pathways of protein folding and unfolding, and mapping partially folded intermediate states, are of utmost importance for our ability to decipher the physiology of misfolding diseases. The generation of maps of folding pathways on the energy landscapes of proteins is the long range goal of our research. Here we propose to address this goal by applying powerful single-molecule fluorescence methodology to study the folding of selected protein systems. We will use fluorescence resonance energy transfer (FRET) to follow conformational dynamics of protein molecules trapped within surface-tethered lipid vesicles, a unique method developed in our lab. Vesicles will be prepared under conditions where molecules have equal folding and unfolding rates, so that single- molecule trajectories will show frequent transitions between conformational states, including partially-folded intermediates. Analysis of trajectories will yield a wealth of information on folding pathways and rates of interconversion between folding intermediates. In addition, studies on freely-diffusing molecules will probe global structural changes of the proteins during folding. We will first study the 62 amino-acid protein L, known to be a two-state folder from bulk experiments. We will verify this observation on the single molecule level, as well as obtain structural information on the denatured state of the protein. We will also study a larger protein, adenylate kinase, which, as we already showed, possesses a very heterogeneous folding energy lansdcape. Intermediates observed during folding of this protein will be carefully mapped. A low-resolution structure of these intermdiates will be obtained by measuring several pairs of labeling sites. In a strong collaboration with Lynne Regan from Yale we will study single-molecule folding of the tetratricopeptide repeat proteins. This family of modular proteins, constructed of 34 amino-acid repeat units, has been suggested to obey a new folding paradigm, based on the classical Ising model, which predicts a large population of partially folded states near the transition midpoint. Single-molecule measurements will probe this intriguing assertion. This study will have impact on our understanding of misfolding diseases, like Alzheimer's and type II diabetes. Our experiments should help us identifying intermediate structures that act as 'weak points' during folding, leading to protein aggregates which are responsible for disease. [unreadable] [unreadable] [unreadable]